Second TC complex from xenorhabdus

ABSTRACT

The subject invention relates to novel  Xenorhabdus  toxin complex (TC) proteins and genes that encode these proteins. More specifically, the subject invention relates to TC genes and proteins obtainable from  Xenorhabdus  strain Xwi.

The present invention relates to a novel toxin complex (TC) proteinobtainable from Xenorhabdus and to novel insecticidal combinationscomprising this protein and other TC proteins.

BACKGROUND OF THE INVENTION

There is a great need for developing genes that can be expressed inplants in order to provide transgenic insecticidal plants thateffectively control various insects. Recently, considerable attentionhas been given to toxin complex (TC) genes, obtainable from a variety oforganisms, including Photorhabdus, Xenorhabdus, Paenibacillus, Serratia,and Pseudomonas.

There has been substantial progress in the cloning of genes encodinginsecticidal toxins from both Photorhabdus luminescens and Xenorhabdusnematophilus. Toxin-complex encoding genes from P. luminescens wereexamined first. See WO 98/08932. Parallel genes were more recentlycloned from X. nematophilus. Morgan et al., Applied and EnvironmentalMicrobiology 2001, 67:20062-69; WO 95/00647 relates to the use ofXenorhabdus protein toxin to control insects, but it does not recognizeorally active toxins. WO 98/08388 relates to orally administeredpesticidal agents from Xenorhabdus. U.S. Pat. No. 6,048,838 relates toprotein toxins/toxin complexes, having oral activity, obtainable fromXenorhabdus species and strains.

Four different toxin complexes (TCs)—Tca, Tcb, Tcc and Tcd—have beenidentified in Photorhabdus spp. Each of these toxin complexes resolvesas either a single or dimeric species on a native agarose gel butresolution on a denaturing gel reveals that each complex consists of arange of species between 25-280 kDa. See WO 97/17432, WO 98/08932, andR. H. ffrench-Constant and Bowen, 57 Cell. Mol. Life Sci. 828-833(2000).

Genomic libraries of P. luminescens were screened with DNA probes andwith monoclonal and/or polyclonal antibodies raised against the toxins.Four tc loci were cloned: tca, tcb, tcc and tcd. The tca locus is aputative operon of three open reading frames (ORFs), tcaA, tcaB, andtcaC, transcribed from the same DNA strand, with a smaller terminal ORF(tcaZ) transcribed in the opposite direction. The tcc locus also iscomprised of three ORFs putatively transcribed in the same direction(tccA, tccB, and tccC). The tcb locus is a single large ORF (tcbA), andthe tcd locus is composed of two ORFs (tcdA and tcdB) ; tcbA and tcdA,each about 7.5 kb, encode large insect toxins. TcdB has some level ofhomology to TcaC. It was determined that many of these gene productswere cleaved by proteases. For example, both TcbA and TcdA are cleavedinto three fragments termed i, ii and iii (e.g. TcbAi, TcbAii andTcbAiii). Products of the tca and tcc ORFs are also cleaved. See WO98/08932 and R. H. french-Constant and D. J. Bowen, Current Opinions inMicrobiology, 1999, 12:284-288.

Bioassays of the Tca toxin complexes revealed them to be highly toxic tofirst instar tomato hornworms (Manduca sexta) when given orally (LD₅₀ of875 ng per square centimeter of artificial diet). R. H. french-Constantand Bowen 1999. Feeding was inhibited at Tca doses as low as 40 ng/cm².Given the high predicted molecular weight of Tca, on a molar basis, P.luminescens toxins are highly active and relatively few molecules appearto be necessary to exert a toxic effect. R. H. ffrench-Constant andBowen, Current Opinions in Microbiology, 1999, 12:284-288.

WO 99/42589 and U.S. Pat. No. 6,281,413 disclose TC ORFs fromPhotorhabdus luminescens. WO 00/30453 and WO 00/42855 disclose TCproteins from Xenorhabdus.

While the exact molecular interactions of the TCs with each other, andtheir mechanism(s) of action, are not currently understood, it is known,for example, that the Tca toxin complex (comprised of TcaA, TcaB, andTcaC) of Photorhabdus is toxic to Manduca sexta. In addition, some TCproteins are known to have “stand alone” insecticidal activity, whileother TC proteins are known to potentiate or enhance the activity of thestand-alone toxins. It is known that the TcdA protein is active, alone,against Manduca sexta, but that TcdB and TccC, together, can be used (inconjunction with TcdA) to greatly enhance the activity of TcdA. TcbA isthe other main, stand-alone toxin from Photorhabdus. The activity ofthis toxin (TcbA) can also be greatly enhanced by TcdB- together withTccC-like proteins.

Photorhabdus Photorhabdus strain W14 TC protein nomenclature Somehomology to: TcaA Toxin C TccA TcaB TccB TcaC TcdB TebA Toxin B TccAToxin D TcdA N terminus TccB TcdA C terminus TccC TcdA Toxin A TccA +TccB or TcaA + TcaB TcdB TcaC

TcaA, TcaB, TccA, and TccB are referred to as “small toxins”, asdistinguished from the “large toxins” TcbA and TcdA. The pair TcaA+TcaBin Toxin C is analogous to the large toxin TcdA in Toxin A; and in thepair TccA+TccB in Toxin D is likewise analogous to the large toxin TcdA.N. R. Waterfield, et al., “The tc genes of Photorhabdus: a growingfamily,” TRENDS IN MICROBIOLOGY, Vol. 9, pp 185-191 (2001).

Some Photorhabdus TC proteins have some level of sequence homology withother Photorhabdus TC proteins. As indicated above, TccA has some levelof homology with the N terminus of TcdA, and TccB has some level ofhomology with the C terminus of TcdA. Furthermore, TcdA is about 280kDa, and TccA together with TccB are of about the same size, ifcombined, as that of TcdA. Though TccA and TccB are much less active onSCR than TcdA, TccA and TccB from Photorhabdus strain W14 are called“Toxin D.” “Toxin A” (TcdA), “Toxin B” (Tcb or TcbA), and “Toxin C”(TcaA and TcaB) are also indicated above.

Furthermore, TcaA has some level of homology with TccA and likewise withthe N terminus of TcdA. Still further, TcaB has some level of homologywith TccB and likewise with the N terminus of TcdA. TcdB has asignificant level of similarity to TcaC.

Relatively recent cloning efforts in Xenorhabdus nematophilus alsoappear to have identified novel insecticidal toxin genes with homologyto the P. luminescens tc loci. See, e.g., WO 98/08388 and Morgan et al,Applied and Environmental Microbiology 2001, 67:20062-69. In R. H.ffrench-Constant and D. J. Bowen Current Opinions in Microbiology, 1999,12:284-288, cosmid clones were screened directly for oral toxicity toanother lepidopteran, Pieris brassicae. One orally toxic cosmid clonewas sequenced. Analysis of the sequence in that cosmid suggested thatthere are five different ORF's with similarity to Photorhabdus tc genes;orf2 and orf5 both have some level of sequence relatedness to both tcbAand tcdA, whereas orf1 is similar to tccB, orf3 is similar to tccC andorf4 is similar to tcaC. Importantly, a number of these predicted ORFsalso share the putative cleavage site documented in P. luminescens,suggesting that active toxins may also be proteolytically processed.

Five typical TC proteins from Xenorhabdus have heretofore beenidentified: XptA1, XptA2, XptB1, XptC1, and XptD1. XptA1 and XptA2 wereknown to have stand-alone toxin activity. The XptA2 protein was known tohave some degree of similarity to the TcdA protein. XptB1 and XptC1 areXenorhabdus potentiators that were known to enhance the activity ofeither (or both) of the XptA toxins. XptD1 was known to have some levelof homology with TccB, and XptC1 was known to have some level ofsimilarity to TcaC. XptB1 has some level of similarity to TccC.

United States Patent Application 20040194164 of Scott B. Bintrim et al.on “Xenorhabdus TC proteins and genes for pest control” discloses a setof novel Xenorhabdus TC proteins and genes obtainable from the Xwistrain of Xenorhabdus nematophilus. It also provides an exochitinaseobtainable from from the Xwi strain of Xenorhabdus nematophilus.

TC proteins and genes have more recently been described from otherinsect-associated bacteria such as Serratia entomophila, an insectpathogen. Waterfield et al., TRENDS in Microbiology, Vol. 9, No. 4,April 2001.

TC proteins and lepidopteran-toxic Cry proteins have very recently beendiscovered in Paenibacillus. See U.S. Ser. No. 60/392,633 (Bintrim etal.), filed Jun. 28, 2002. Bacteria of the genus Paenibacillus aredistinguishable from other bacteria by distinctive rRNA and phenotypiccharacteristics (C. Ash et al. (1993), “Molecular identification of rRNAgroup 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probetest: Proposal for the creation of a new genus Paenibacillus,” AntonieVan Leeuwenhoek 64:253-260). Some species in this genus are known to bepathogenic to honeybees (Paenibacillus larvae) and to scarab beetlegrubs (P. popilliae and P. lentimorbus.) P. larvae, P. popilliae, and P.lentimorbus are considered obligate insect pathogens involved with milkydisease of scarab beetles (D. P. Stahly et al. (1992), “The genusBacillus: insect pathogens,” p. 1697-1745, In A. Balows et al., ed., TheProcaryotes, 2^(nd) Ed., Vol. 2, Springer-Verlag, New York, N.Y.).

Although some Xenorhabdus TC proteins have been found to “correspond”(have a similar function and some level of sequence homology) to some ofthe Photorhabdus TC proteins, the “corresponding” proteins share onlyabout 40% (approximately) sequence identity with each other. This isalso true for the more recently discovered TC proteins fromPaenibacillus (those proteins and that discovery are the subject ofco-pending U.S. Ser. No. 60/392,633).

Some TC proteins have stand alone insecticidal activity, but it is knownthat this activity can be enhanced when such proteins are used incombination with other TC proteins. In this regard, three relevantclasses of TC proteins have heretofore been identified: Class Aproteins, Class B proteins, and Class C proteins. A protein belonging toone of these classes is referred to hereinafter as a “Protein A”, a“Protein B”, or a “Protein C”. Proteins are assigned to Class A, ClassB, or Class C based on sequence similarity, as discussed in greaterdetail hereinafter. Class A proteins have stand alone insecticidalactivity. Typical examples of Class A proteins are the “large toxins”TcdA, TcdA2, TcdA4, and TcBA from Photorhabdus luminescens, XptA1_(Xwi)and XptA2_(Xwi) from Xenorhabdus nematophilus, and SepA from Serratiaentomophila. Class A proteins should also be understood to include thesmall toxin pairs that correspond to the large toxins, e.g. TcaA+TcaB inToxin C and TccA+TccB in Toxin D. Class B and Class C proteins lacksignificant stand alone insecticidal activity, and have been referred toas potentiators. Typical Class B proteins are TcdB1, TcdB2, TcaC fromPhotorhabdus luminescens, XptC1_(Xwi) Xenorhabdus nematophilus,PptB1₁₅₂₉ from Paenibacilluss spp., and SepB from Serratia entomophila.Typical Class C proteins are TccC2 from Photorhabdus luminescens,XptB1_(Xwi) from Xenorhabdus nematophilus, and XptC1_(Xb) fromXenorhabdus bovienii. It has repeatedly been found that use of a ProteinA in combination with a Protein B and a Protein C substantially enhancesinsecticidal activity over that obtained with the Protein A alone.

United States Patent Application Publication 2004/0208907 demonstratesthat the activity of a Protein A can be potentiated by a Protein B andProtein C even if the Protein B and/or Protein C originates from anentirely distinct species from the one that produces the Protein A.

In light of concerns about insects developing resistance to a givenpesticidal toxin, and in light of other concerns—some of which arediscussed above, there is a continuing need for the discovery of newinsecticidal toxins and other proteins that can be used to controlinsects.

BRIEF SUMMARY OF THE INVENTION

The present invention provides the novel toxin complex (TC) proteincomplex XTC-2 comprising XptB1 (SEQ ID NO:11), XptC1 (SEQ ID NO:10),XptD1 (SEQ ID NO:2), XptE1 (SEQ ID NO:4), and an exochitinase of SEQ IDNO:6.

The invention also provides a novel toxin complex (TC) protein,XptE1_(Xwi) and DNA sequences encoding it. XptE1_(Xwi) complements thepreviously known XptD1_(Xwi) in a similar fashion to the pair TcaA+TcaB.That is, the XptD1_(Xwi)+XptE1_(Xwi) pair provides the equivalent of a“large toxin” lass A protein.

The invention also provides insecticidal compositions comprisingXptE1_(Xwi), and methods of controlling insects using it.

More specifically, the invention provides an isolated protein that hastoxin activity against an insect, wherein said protein has at least 50%sequence identity with the amino acid sequence of SEQ IDNO:4(XptE1_(Xwi)).

The invention also provides insectidical compositions comprising, incombination, a protein having the amino acid sequence of SEQ ID NO:2(XptD1_(Xwi)), a protein having the amino acid sequence of SEQ ID NO:4(XptE1_(Xwi)), a Protein B, and a Protein C, wherein

said Protein B is a 130-180 kda toxin complex potentiator having anamino acid sequence at least 40% identical to a sequence selected fromthe group consisting of:

SEQ ID NO:7 (TcdB1), SEQ ID NO:8 (TcdB2), SEQ ID NO:9 (TcaC), SEQ IDNO:10 (XptC1_(Xwi)), SEQ ID NO:11 (XptB1_(Xb)), SEQ ID NO:12(PptB1(orf5)), and SEQ ID NO:13 (SepB); and

said Protein C is a 90-120 kDa toxin complex potentiator having an aminoacid sequence at least 35% identical to a sequence selected from thegroup consisting of:

SEQ ID NO:14 (TccC1), SEQ ID NO:15 (TccC2), SEQ ID NO:16 (TccC3), SEQ IDNO:17 (TccC4), SEQ ID NO:18 (TccC5), SEQ ID NO:19 (XptB1_(Xwi)), SEQ IDNO:20 (XptC1_(Xb)), SEQ ID NO:21 (PptC1 (orf 6 long)), SEQ ID NO:22(PptC1 (orf 6 short)), SEQ ID NO:23 (SepC).

FIG. 1 is a map of Cosmid 8C3 (Cosmid 8C3 was provided by HRI). Thecomplete sequences of xptA1, xptA2, xptB1, xptC1 and the exochitinasegenes were obtained from this cosmid. Note that the xptE1 gene ismissing completely from this cosmid, but was obtained from XTC-2.

FIG. 2 shows bar graphs with bioassay data for XTC-2.

BRIEF DESCRIPTION OF THE SEQUENCES

-   SEQ ID NO:1 is the DNA sequence for xptD1_(Xwi).-   SEQ ID NO:2 is the amino acid sequence for XptD1_(Xwi).-   SEQ ID NO:3 is the DNA sequence for xptE1_(Xwi).-   SEQ ID NO:4 is the amino acid sequence for XptE1_(Xwi).-   SEQ ID NO:5 is the DNA sequence for the exochitinase from Xwi.-   SEQ ID NO:6 is the amino acid sequence for the exochitinase from    Xwi.-   SEQ ID NO:7 is the amino acid sequence for TcdB1.-   SEQ ID NO:8 is the amino acid sequence for TcdB2.-   SEQ ID NO:9 is the gene and amino acid sequence for TcaC from    GENBANK Accesion No. AF346497.1.-   SEQ ID NO:10 is the amino acid sequence for XptC1_(Xwi) (1,493 amino    acids).-   SEQ ID NO:11 is the amino acid sequence for XptB1_(Xb) (1506 amino    acids).-   SEQ ID NO:12 is the amino acid sequence for PptB1₁₅₂₉ (Paenibacillus    ORF5) (1445 amino acids).-   SEQ ID NO:13 is the amino acid sequence for the SepC protein (1428    amino acids).-   SEQ ID NO:14 is the gene and protein sequence for TccC1 from GENBANK    Accession No. AAC38630.1.-   SEQ ID NO:15 is the amino acid sequence for TccC2 from GENBANK    Accession No. AAL18492.-   SEQ ID NO:16 is the amino acid sequence for TccC3.-   SEQ ID NO:17 is the amino acid sequence for TccC4_(W-14).-   SEQ ID NO:18 is the gene and amino acid sequence for TccC5 from    GENBANK Accession No. AF346500.2.-   SEQ ID NO:196 is the amino acid sequence for XptB1_(Xwi).-   SEQ ID NO:20 is the amino acid sequence for XptC1_(Xb), (962 amino    acids).-   SEQ ID NO:21 is an alternate (long) amino acid sequence for    PptC1_(1529L).-   SEQ ID NO:22 is an alternate (short) amino acid sequence for    PptC1_(1529S).-   SEQ ID NO:23 is the amino acid sequence for the Sep C protein.

DEFINITIONS

Isolated polynucleotides and isolated proteins. As used herein,reference to “isolated” polynucleotides and/or “purified” toxins refersto these molecules when they are not associated with the other moleculeswith which they would be found in nature. Thus, reference to “isolated”and/or “purified” signifies the involvement of the “hand of man” asdescribed herein. For example, a bacterial toxin “gene” of the subjectinvention put into a plant for expression is an “isolatedpolynucleotide.” Likewise, a Xenorhabdus protein, exemplified herein,produced by a plant is an “isolated protein.”

Toxin activity against an insect. By toxin activity against an insect itis meant that a protein function as an orally active insect controlagent (alone or in combination with other proteins), as demonstrated byits ability to disrupt or deter insect activity, growth, and/or feeding.Toxin activity may or may not cause death of the insect. When an insectcomes into contact with an effective amount of a “toxin” of the subjectinvention delivered via transgenic plant expression, formulated proteincomposition(s), sprayable protein composition(s), a bait matrix or otherdelivery system, the results are typically death of the insect,inhibition of the growth and/or proliferation of the insect, and/orprevention of the insects from feeding upon the source (preferably atransgenic plant) that makes the toxins available to the insects.Complete lethality to feeding insects is preferred but is not requiredto achieve functional activity. If an insect avoids the toxin or ceasesfeeding, that avoidance will be useful in some applications, even if theeffects are sublethal or lethality is delayed or indirect. For example,if insect resistant transgenic plants are desired, the reluctance ofinsects to feed on the plants is as useful as lethal toxicity to theinsects because the ultimate objective is avoiding insect-induced plantdamage.

Sequence identity. Unless otherwise specified, as used herein percentsequence identity and/or similarity of two nucleic acids is determinedusing the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad.Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993), Proc.Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (1990), J. Mol.Biol. 215:402-410. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST is used as described in Altschulet al (1997), Nucl. Acids Res. 25:3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) are used. See NCBI/NIH website. The scores can alsobe calculated using the methods and algorithms of Crickmore et al. asdescribed in the Background section, above.

Hybridizes under stringent conditions. As used herein “stringent”conditions for hybridization refers to conditions which achieve thesame, or about the same, degree of specificity of hybridization as theconditions employed by the current applicants. Specifically,hybridization of immobilized DNA on Southern blots with ³²P-labeledgene-specific probes was performed by standard methods (see, e.g.,Maniatis, T., E. F. Fritsch, J. Sambrook [1982] Molecular Cloning. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). In general, hybridization and subsequent washes were carried outunder conditions that allowed for detection of target sequences. Fordouble-stranded DNA gene probes, hybridization was carried out overnightat 20-25E C below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Themelting temperature is described by the following formula (Beltz, G. A.,K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983]Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] AcademicPress, New York 100:266-285):Tm=81.5E C+16.6 Log[Na+]+0.41 (% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1× SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm-20EC for 15 minutes in 0.2× SSPE, 0.1% SDS (moderatestringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20EC below the melting temperature (Tm) of the hybrid in 6× SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula:Tm (E C)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura,and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes,D. D. Brown [ed.], Academic Press, New York, 23:683-693).

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1× SSPE, 0.1% SDS (lowstringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1× SSPE,0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment>70 or so bases in length, the followingconditions can be used:

Low: 1 or 2x SSPE, room temperature Low: 1 or 2x SSPE, 42E C. Moderate:0.2x or 1x SSPE, 65E C. High: 0.1x SSPE, 65E C.

Protein B. Protein B refers to a 130-180 kDa toxin complex potentiatorhaving an amino acid sequence at least 40% identical to a sequenceselected from the group consisting of:

-   SEQ ID NO:7 (TcdB1), SEQ ID NO:8 (TcdB2), SEQ ID NO:9 (TcaC), SEQ ID    NO:10 (XptC1_(Xwi)), SEQ ID NO:11 (XptB1_(Xb)), SEQ ID NO:12    (PptBp1(orf5)), and SEQ ID NO:13(SepB).

Protein C. Protein C refers to a 90-120 kDa toxin complex potentiatorhaving an amino acid sequence at least 35% identical to a sequenceselected from the group consisting of:

-   SEQ ID NO:14 (TccC1), SEQ ID NO:15 (TccC2), SEQ ID NO:16 (TccC3),    SEQ ID NO:17 (TccC4), SEQ ID NO:18(TccC5), SEQ ID NO:19    (XptB1_(Xwi)), SEQ ID NO:20(XptC1_(Xb)), SEQ ID NO: 21 (PptC1(orf 6    long)), SEQ ID NO:22 (PptC1(orf 6 short)), SEQ ID NO:23 (SepC).

Potentiator. Potentiator protein refers to a TC “Protein B” or “ProteinC.”

Associating a toxic protein with a material to be protected can beachieved by, e.g. applying the toxic protein to the material, admixingit with the material, and, in the case of transgenic plant material, bycausing the plant cells to express the toxic protein.

Effective amount of a toxic protein means an amount that provides toxicactivity against an insect.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

We describe here the purification and characterization of a novellepidopertan-active protein toxin complex termed Xenorhabdus ToxinComplex 2 (XTC-2) isolated from Xenorhabdus nematophilus (Xwi strain).We determined XTC-2 to be comprised of five different proteins,XptC1_(Xwi) (a 170 kDa “B” type protein), XptD1_(Xwi) (a 140 kDaprotein), XptE1_(Xwi) (a 130 kDa protein), XptB1_(Xwi) (112 kDa “C” typeprotein) and an exochitinase. The molecular weight of XTC-2 wasestimated to be between 1,150-1,300 kDa based upon migration on nativepolyacrylamide gel electrophoresis (PAGE) and elution volume from sizeexclusion chromatography. Based upon its estimated molecular weight andthe size of the individual protein subunits, we determined that multiplecopies of the subunits are most likely present in the complete nativeprotein. When fed to insects in a top load diet bioassay, XTC-2 resultsin inhibition of larval growth, with fifty percent inhibition of growth(GI₅₀) occurring with approximately 1 ng/cm² of applied material whentested against corn earworm larvae (CEW, Helicoverpa zea,) and 10-70ng/cm² vs tobacco budworm larvae (TBW, Heliothis virescens). The toxincomplex has weak activity against beet army worm (BAW, Spodopteraexigua), and did not inhibit growth of southern corn rootworm larvae(SCR, Diahrotica undecimpunctata howardi) at protein concentrations ashigh as 1,000 ng/cm². This limited spectrum of insect bioassaysdemonstrates that XTC-2 has activity against lepidopteran insects, butfurther bioassays would be needed to fully characterize its spectrum ofinsect activity.

Methods

Materials. Aqueous solutions were prepared using Milli-Q purified water.All solvents used were HPLC grade. All other chemicals were analyticalgrade from commercial supply houses. Bovine serum albumin (BSA) FractionV (cat. #A7906), and the general protease inhibitors for bacterial celllysates (cat. #P8465) were both from Sigma Chemical, (St. Louis, Mo.).Native molecular weight protein markers were from Amersham (Piscataway,N.J., cat. #17-0445-01). Protein liquid chromatography was performed onan AKTA purifier 900 chromatography system.

Protein Purification. Cell pellets obtained from a 2 liter culture afterovernight incubation of the Xenhorabdus nematophihis bacteria wereprovided by Scott Bintrim, and typically stored frozen at −20° C. forless than two weeks. Frozen cells were suspended in 250 ml of 50 mMTris-HCl pH 8.0, 0.10 M NaCl, 1 mM DTT, 10% glycerol and lysozyme (0.6mg/ml). A small amount of glass beads (0.5 mm), was added and thesolution gently shaken to facilitate suspension. The cells were thendisrupted in approx. 50 ml batches by sonicating them at maximum outputpower two times for 30 seconds, keeping the lysate cold using an icebath. The broken cells were then centrifuged at 48,000×g for 60 min. at4° C. The supernatant was collected, a bacterial protease inhibitorcocktail added (2.0 ml), and the solution dialyzed against 25 mMTris-HCl, pH 8.0 overnight. The protein was then loaded onto a Qsepharose XL (1.6×10 cm) anion exchange column. Bound proteins wereeluted using a linear 0 to 1M NaCl gradient in 10 column volumes at aflow rate of 5 ml/min and collecting in 5 ml fractions. The highmolecular weight toxin complexes eluted in the early fractions. Thesefractions were combined and concentrated to 2 ml using a 100 kDa filterconcentrator. The concentrated proteins were then loaded onto a superose200 size exclusion column (1.6×60 cm), using 50 mM Tris-HCl, 100 mMNaCl, 5% glycerol, 0.05% Tween-20, pH 8.0 at a flow rate of 1.0 m/min.The large molecular weight proteins eluting from the column, werecombined, brought to 1.5 M ammonium sulfate concentration and loadedonto a phenyl superose (0.5×5 cm) hydrophobic-interaction column.Proteins were eluted using a decreasing linear gradient of 1.5 to 0 Mammonium sulfate in 25 mM Tris-HCl, pH 8.0 over 20 column volumes at aflow rate of 1 ml/min. The toxin complexes eluted together as a broadpeak at low salt concentration. The protein was dialyzed overnightagainst 25 mM Tris-HCl, and loaded onto a high resolution MonoQ (0.5×5cm) anion exchange column. The two separate toxin complexes wereresolved with baseline resolution using a linear gradient of 0 to IMNaCl in 25 mM Tris-HCl obtained in 20 column volumes. The proteins wereidentified by N-terminal amino acid sequencing and Matrix-Assisted LaserDesorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometryanalysis as described below.

Gel electrophoresis. Purification was monitored by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing anddenaturing conditions by method of Laemmli (Laemmli, 1970). Variousquantities of protein were loaded into wells of a 4-20% tris-glycinepolyacrylamide gel (Zaxis, Hudson, Ohio) and separated by applying 170volts of electricity for 90 minutes. Detection of the separated proteinbands was achieved by staining the gel with Coomassie brilliant blueR-250 (BioRad, Hercules, Calif.) for one hour, then destaining firstwith a solution of 45% methanol and 10% acetic acid, then with asolution of 5% methanol and 7% acetic acid. The gels were imaged andanalyzed using a BioRad Fluro-S Multi Imager™. Relative molecular weightof the protein bands was determined by including a sample of BenchMark™Protein Ladder (Life Technologies, Rockville, Md.) in one well of thegel.

Proteolytic digestion. SDS gel slices containing Coomassie blue stainedbands were removed from the gel and placed into a siliconized Eppendorfmicrocentrifuge tubes. The gel slices were destained with 50%acetonitrile in 25 mM NH₄HCO₃ until the slices became clear and thesolvent removed using a Speed-Vac (Savant Instruments, Holbrook, N.Y.).The dried gel slices were crushed and enough sequencing grade trypsin at12.5 μg/ml (Roche Diagnostics, Indianapolis, Ind.) was added to coverthe gel pieces. After 30 min incubation at room temperature the excesstrypsin was removed and replaced with 25 mM NH₄HCO₃. The digestion wasallowed to proceed for 4 hr at 37° C. The digested peptides were thenextracted by shaking for one hour with a solution of 50% acetonitrile in0.5% trifluoro acetic acid (TFA, Sigma). After brief centrifugation topellet the gel pieces, the supernatant containing the peptides wastransferred to a fresh tube and dried in a Speed-Vac. The peptides werethen suspended in 6 μL of 0.1% TFA, absorbed to a C₁₈ ZipTip resin(Millipore, Bedford, Mass.) and eluted with 75% acetonitrile/0.1% TFA.The eluent was stored at −20° C. freezer until MALDI-TOF mass spectralanalysis.

Fragment derivatization. To facilitate MALDI-Post Source Decay (PSD)sequencing the peptides were modified using Ettan™ CAF™ Sequencing Kit(Amersham). This modification improves fragmentation efficiency atpeptide bonds and results in the formation of only positively charged yions which become separated in the MALDI reflection. Briefly thepeptides were absorbed onto a C18 Ziptip resin. The lysine residues atC-terminus were protected by converting to homoarginine usingO-methylisourea-hydrogen sulfate and incubating overnight. After washingthe ziptip with water to remove the quanidination reagent, theN-terminal of the peptides were sulfonated with provided CAF reagentsolution for at least three min. The ziptip was washed and thederivatized peptides were eluted with 80% acetonitrile for MALDI-PSDsequencing.

Mass Spectrometry Analysis. The extracted peptides were analyzed usingMALDI-TOF mass spectrometry. The instrument used was a Voyager DE-STRMALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, Mass.).The instrument utilizes a 337 nm nitrogen laser for thedesorption/ionization event and a 3.0 meter reflectron time-of-flighttube.

To generate peptide mass fingerprints the samples derived above werespotted onto a MALDI stainless steel plate in a 1:1 ratio of 0.5 μL ofsample with 0.5 μL of matrix mixed on the plate using the dried dropletspotting technique (air dried). The matrix was a saturated solution ofα-cyano-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% TFA.External calibration was performed by using a solution of CalibrationMix 2 (PE Biosystems, Foster City, Calif.). Internal calibration wasperformed using, if detected, the autolytic trypsin peak at m/z 842.50,1045.56, or 2211.10. Acquired spectra were de-isotoped and peptide massfingerprints (PMF) tables were exported for database searching.

Chemically modified peptides were used for MALDI-PSD sequencing. Theinstrument was first calibrated with an angiotensin peptide at 1296 Da.Calibration for PSD fragments was performed using the fragmentedangiotensin peptide as described by the instrument manual. MALDI-MS wasfirst performed on the samples in both positive and negative modes togenerate mass fingerprints. From the fingerprints at least five peptideswith high intensities were selected for MALDI-PSD analysis. PSDfragmentation and detection was performed by incrementally increasinglaser intensity by 50 V and tilting the reflectron mirror ratio from 1.0to 0.2. The fragment spectra from different mirror ratios were stitchedtogether by instrument control software. The mass differences betweenneighboring peaks were calculated and their corresponding amino acid wasdetermined from an amino acid mass table.

Edman N-terminal amino acid sequencing. After SDS PAGE separation,proteins were transferred to PVDF membrane (Bio-Rad) using standardprocedure (100V for one hr). The membrane was stained briefly withCoomassie blue, the protein bands removed, dried and sent for N-terminalsequencing. N-terminal sequencing was performed with a Procise 494(Applied Biosystem, Freemont, Mass.) using a standard protocol providedby the manufacture. Sequence was determined by accompanied softwareSequence Pro Ver. 1.0 and then manually corrected for erroneous sequenceassignments.

Database searching and protein identification. Proteomics search engineMascot (London, UK) was used for database searching and proteinidentification. A proprietary toxin database was constructed bycompiling all the known (public and private) Photorhabdus andXenorhabdus toxin related protein and DNA translation sequences. PMFtables were pasted in the Mascot search form and the search performedagainst both the toxin database and NCBI non-redundant protein database.No molecular weight and pI restrictions were applied during the search,nor during the numerous modifications. The mass error tolerance was setat 0.1 Da and missed cleavage was set at 1.

Insect bioassays. Corn earworm (CEW, Helicoverpa zea) used in thesestudies were supplied as eggs by the insectary at North Carolina StateUniversity. Tobacco budworm (TBW, Heliothis virescens) and beet armyworm(BAW, Spodoptera exigua) eggs were obtained from the insectary of DowAgroSciences. Southern corn rootworm eggs (SCR, Diabroticaundecimpunctata howardi) were supplied by FrenchAg Research, Lamberton,Minn., or Crop Characteristics, Inc., Farmington, Minn. The eggs werewashed and held at 24° C. and 50% RH until they hatched. The artificialdiet consists of 2-4% powdery solids such as soy flour, yeast, wheatgerm, casein, sugar, vitamins, and cholesterol suspended in a 1.0-2.0%dissolved agar in water matrix. For bioassay, toxin protein complexeswere diluted in ten-fold increments into 10 mM sodium phosphate buffer,pH 7.0 to concentrations ranging from 1 to 1000 ng toxin per cm² thenapplied to the surface of the artificial diet. Each concentration wasassayed separately with 8 replications by placing newly emerged neonatesonto the treated diet and holding the test at 28° C. for five days. Theweights of the larvae were measured at the end of the time period inaddition to recording mortality or stunting of the insects. Dead larvaewere scored as zero weight. GI₅₀ was determined using the measuredweights of the insects and the Trimmed Spearman Karber statisticalmethod (Hamilton et al., 1977).

Results

Toxin Complex Purification and Characterization. Initial anionchromatography of the cell lysate from Xenorhabdus nematophilus resultedin poor resolution but captured the majority of the high molecularweight proteins, which eluted between 100-200 mM salt concentrations.Subsequent size exclusion chromatography isolated a well-resolved highmolecular weight fraction eluting at 40 ml very near the void volume ofthe column. This fraction was further resolved into two broad andsymmetrical peaks in an approximate 7:1 ratio by hydrophobic-interactionchromatography. The high molecular weight proteins are located in thelarger peak eluting at approximately 0.4 M ammonium sulfate and wereresolved into two separate peaks using a high resolution MonoQ anionexchange column. The two peaks elute between 35-45 mS/cm conductance.When analyzed by SDS-PAGE, the first peak was determined to consist ofthree proteins. Using a calibration curve generated from separation ofthe Benchmark Ladder, these three proteins have apparent molecularweights of 280, 172, and 85 kDa. The molecular weight of these proteinscorrespond to those found in Xeno Toxin Complex 1 (XTC-1) previouslyidentified (Morgan et al., 2001; Sergeant et al., 2002) composed ofproteins XptA2_(Xwi), XptC1_(Xwi) and XptB1_(Xwi), respectively.Confirmation of the identity of these proteins was done by MALDI-TOFanalysis of each of the protein bands.

The second peak from the MonoQ anion exchange column termed as XenoToxin Complex 2 (XTC-2), is composed of at least 4 proteins asdetermined by SDS-PAGE. Based upon migration on SDS PAGE under reducingconditions, the apparent molecular weights of the four protein bandscorrespond to 172, 153, 131, and 85 kDa. When analyzed by native PAGEusing a 3-8% tris acetate gel, a single high molecular weight proteinband is obtained, showing that the individual proteins resolved bySDS-PAGE form a single protein complex under native conditions. Usingfour molecular weight standards in the native gel, we generated astandard curve for migration of proteins under native conditions. Basedupon the relative migration of XTC-2 by PAGE under native conditions andcompared to known standards, it has an apparent molecular weight ofabout 1,300 kDa. Note that due to the very high molecular weight ofXTC-2, this value was obtained by extrapolation from the calibrationcurve. The high molecular weight of XTC-2 indicates that the complex ismost likely not only composed of at least the four different proteinsresolved by SDS-PAGE, but that multiple copies of the different proteinsare most likely included as part of the complex.

When loaded on to a calibrated Superose 200 1×30 cm size exclusioncolumn, XTC-2 eluted as a single symmetrical peak, very close to thevoid volume of the column. Three separate injections of XTC-2 were madeusing the calibrated superose 200 1×30 cm column, resulting in elutionof the toxin complex at 8.13, 8.19 and 8.02 ml (average=8.11 ml). Usinga calibration curve for this size exclusion column, this elution volumecorresponded to an apparent molecular weight of 1,150 kDa (range1,100-1,200 kDa). This value is close to the value obtained by nativePAGE analysis (1,300 kDa), and further demonstrates that XTC-2 is mostlikely composed of multiple copies of the different subunits that can beseparated by SDS-PAGE under denaturing conditions.

Densitometric analysis of the SDS-PAGE gel indicates that the differentprotein bands comprising the XTC-2 are not present at equalconcentrations or molar ratios (Table 1). XptC1_(Xwi) is present in thelowest amount, based upon density of staining by coomassie blue dye.Given that this is the largest protein (172 kDa) of the five proteinscomprising XTC-2, on a molar basis the content of this protein is verylow relative to the molar amount of the other proteins subunitscomprising the total toxin. Since the exochintinase and XptB1_(Xwi) waspoorly resolved on this gel, we did not determine the relativeproportions of these two different proteins comprising the entire ToxinComplex.

Protein Identification. The proteins in the complexes were resolved bySDS-PAGE. From previous work it is known that XTC-1 is composed of four280 kDa toxin subunits (named XptA2_(Xwi)) and one XptC1_(Xwi) and oneXptB1_(Xwi) protein. The components of XTC-1 were confirmed as expectedas XptA2_(Xwi), XptC1_(Xwi) and XptB1_(Xwi,). Although the particularpreparation of XTC-2 illustrated here contains a high molecular weightprotein (˜280 kDa) possibly corresponding to XptA2_(Xwi), in subsequentsteps this protein was removed and thus is not part of XTC-2. Two veryfaint proteins at about 32 kDa were evaluated by MALDI and appear to bebreakdown products of the XptD1_(Xwi) and XptB1_(Xwi) proteins (data notshown). These data suggest that toxin complex 2 is composed of at least4 peptides.

To identify these peptides the protein bands were either blotted ontoPVDF membrane for N-terminal sequencing, or analyzed with MALDI-TOF.Initial attempt to identify the peptides in lane 3 using MALDI peptidemass fingerprints (PMF) failed to produce positive hits due to lack ofprotein sequences in our proprietary toxin database. We subsequentlysearched for all the Photorhabdus and Xenorhabdus sequences from ourprevious sequencing efforts and from publicly available database (NCBInon-redundant database and Swiss-Prot database). Obtained DNA sequenceswere translated into amino acid sequences. This new set of amino acidsequences was then updated to our toxin database. The database searchingwas repeated against the new toxin database and positive hits with highconfidences were returned for all bands in XTC-2. The top band (MW ˜172kDa) was found to be highly similar to (>95%), if not identical toXptC1_(Xwi), a protein also found in XTC-1. Forty matching trypticfragments cover 35% of amino acids in the sequence, yielding a highlysignificant Mowse score of 290, (a score greater than 40 is significantat the 0.05 level). The second band (MW ˜153) was highly similar to, ifnot identical to, XptD1_(Xwi). Forty four matching fragments cover 44%of amino acids in the sequence, producing a Mowse score of 270. Thesignificant hit (Mowse score 121) for the third band (˜131 kDa)corresponds to Xwi XptE1_(Xwi). However, calculated MW from theXptE1_(Xwi) sequence at hand is only 57 kD, significantly smaller than131 kD as indicated by SDS PAGE, indicating the sequence in the toxindatabase is incomplete. The fourth band (˜85 kDa) was found to containat least two peptides. This was confirmed upon close inspection of thegel that shows two very slightly resolved bands (not very obvious on thedigital image). Database searching using mix of the PMFs revealed thatone protein is highly similar to, if not identical to Xeno XptB1_(Xwi)(Mowse score 111, coverage 24% and matching peptides 18), the othermatches with a Xwi exochitinase (Mowse score 195, coverage 52%, andmatching peptides 21). The detail database search results are attachedas appendices.

To confirm the identities of these proteins, they were also subjected toN-terminal and internal sequencing. The N-terminal sequence of band 1,determined by Edman degradation, corresponded to N-terminus ofXptC1_(Xwi). The N-terminal sequence of band 2 was identical toN-terminus of XptD1_(Xwi).

At least five fragments from each of bands corresponding to XptE1_(Xwi),XptD1_(Xwi) and XptB1_(Xwi) were selected for MALDI-PSD analysis. Allthe internal fragment sequences matched with their corresponding proteinsequences as predicted by MALDI PMF data, indicating positiveidentification of these 5 proteins.

It is clear that this toxin complex 2 is formed from 5 proteins, namelyXptC1_(Xwi) XptE1_(Xwi), XptD1_(Xwi), XptB1_(Xwi)and a putativeexochitinase. By inspecting Xwi cosmids that were previouslycharacterized, we found that all or at least part of the genes encodingthese 5 proteins were located on cosmid 8C3 where the toxin genes(XptA2_(Xwi), and XptA1_(Xwi)) of XTC-1 are also located (FIG. 1) Inthis cosmid the XptE1_(Xwi) gene was truncated, which explains thediscrepancy of the 131 kd protein (XptE1_(Xwi)) matching the 57 kd genethat was obtained from this cosmid. It also explains our inability toidentify XTC-2 from screening the cosmid library for bioactivity, sincethe only four complete genes of the five toxin genes were present.

It is not unusual to find toxin complex and auxiliary genes clusteredtogether in the genome. In cosmid 8c3, there are two large operonscomposed of toxin-like genes. The first one contains a truncatedxptE1_(Xwi) gene, a xptD1_(Xwi) gene (which together would form a toxinA like complex), an exochitanase gene and a gene for xptA1_(Xwi). Thestructure of the DNA sequence of the upstream region of the exochitanaseregion is revealing. Its ribosome binding site is sequestered in a largestem loop structure that would prevent this gene from being transcribedexcept as part of the operon. Of course, in the genomic sequencexptE1_(Xwi) would not be truncated. We do not know if xptE1_(Xwi) is thefirst gene in that operon or whether there are more toxin like genesupstream from it. The second operon which is transcribed in the oppositeorientation contains a toxin A gene (xptA2_(Xwi)), a xptC1_(Xwi) geneand a xptB1_(Xwi) gene. These two operons are in extremely closeproximity, the two ends being within a few hundred bases of each other.While the second operon contains all the genes necessary for toxicity,the first operon does not. Toxin gene clustering, as seen in this andother cases, would ensure that genetic recombination would not separatethe first toxin gene operon from the second since the second containsgenes essential for the generation of an active complex from the first.

Bioactivity. Highly purified preparations of XTC-1 and XTC-2 werebioassayed against CEW, TBW, BAW and SCR larvae. Both complexesexhibited a similar spectrum of activity against these four insects.Neither complex was active against SCR larvae when tested at a dietconcentration of up to 1 μg/cm². (data not shown). Both complexes,however, were very active against CEW and TBW, but principally againstCEW larvae. The primary effect of these toxin complexes against thesetwo insects was stunting, but at the highest concentrations tested (1μg/cm²), some death of the larvae was observed. LC-50 values were >1μg/cm² for both of these toxins against these insects. Bioassay data arepresented in the bar graphs shown in FIG. 2. Against CEW, XTC-1 caused50% inhibition of growth (GI₅₀) at between 0.001-0.01 μg/cm², whereasXTC-2 also caused significant stunting of the CEW larvae, but the GI₅₀was in the range of 0.01-0.1 μg/cm². Thus, both toxin complexes arehighly potent against CEW, but XTC-1 is about 10-fold more active thanXTC-2. A similar profile of toxicity is seen with TBW larvae, with XTC-1being more active than XTC-2. Fifty percent growth inhibition of TBWlarvae was observed at less than 10 ng/cm² for XTC-1, whereas XTC-2required about 100 ng/cm². Against BAW larvae, XTC-1 was only moderatelyactive, causing 50 percent growth inhibition at concentrations between0.1 to 1 μg/cm², whereas XTC-2 required more than 1 μg/cm² to inhibitgrowth by 50 percent. With that said, XTC-2 is still very activebiologically and could be of high value for use in imparting insectresistance into plants as it is further understood and the activity ofeach individual protein component is better characterized.

Discussion

This experiment demonstrates the first discovery and characterization ofa second complete toxin complex (XTC-2) found in the Xwi strain ofXenorhabdus nematophihis. Using an active cosmid 8C3 prepared by DowAgroSciences scientists, and from data provided by researchers at HRIusing a different cosmid (cHRIM1), scientists at both institutions wereable to identify the original toxin complex (XTC-1) that consisted ofthree proteins, XptA2_(Xwi), XptC1_(Xwi) and XptB1_(Xwi). The 8C3 cosmidcontains additional genes with unknown function. Some of these genesencode for proteins that we have purified and identified in XTC-2. Theseinclude genes encoding XptC1_(Xwi), XptB1_(Xwi) and the exochitinase.However, parts of the genes encoding the XptD1_(Xwi) and XptE1_(Xwi)proteins were either truncated or abscent in the cHRIM1 cosmid. Anotherprotein belonging to XTC-2 (XptE1_(Xwi)) was missing from the cHRIM1cosmid. The cHRIM1 cosmid also contained an exochitinase gene directlydownstream of the xptD1Xwi gene that was considered not to contribute tobioactivity based on gene inactivation studies. The lack of the completeset of proteins comprising XTC-2 in the cHRIM1 cosmid probably resultedin the poor level of biological activity seen for this cosmid and thusmost likely prevented these researchers from identifying these proteinsas part of an active protein complex. We now have a more completepicture of the genes in cosmid 8C3 and a better understanding of theirfunction.

The XTC-2 is a very large protein complex, estimated at between1,150-1,300 kDa, depending on the method utilized to determine proteinsize. It is composed of at least five different protein subunits. Someof these subunits must be present in multiple copies in the completetoxin complex, since the sum of the molecular weights of the individualcomponents (626 kDa) equals only about half of the molecular weight ofXTC-2. The stochiometry of XTC-1 is 4:1:1, for the XptA2_(Xwi),XptC1_(Xwi) and XptB1_(Xwi) proteins respectively 1. This toxin complexdoes not have an exochitinase associated with it. Given the homologybetween XptE1_(Xwi) and XptD1_(Xwi) proteins with XptA2_(Xwi), it isreasonable to assume that these two proteins represent gene cleavageproducts, that when put together, organize into a protein of similarstructure as XptA2_(Xwi). Thus, XptE1_(Xwi)+XptD1_(Xwi) together couldbe equivalent to one XptA2_(Xwi) protein. Thus, if XTC-1 contains fourXptA2_(Xwi) subunits, then by inference we could assume that XTC-2contains four XptE1_(Xwi) and four XptD1_(Xwi) subunits. This would addup to a combined molecular weight of 1,136 kDa, which is still lowerthan the estimated molecular weight of the entire XTC-2. The addition ofa single XptC1_(Xwi), XptB1_(Xwi), and exochitinase protein would add342 kDa to the weight of the complex, resulting in a overall size of1,478 kDa, which is about 13% larger than the estimated size of thecomplete toxin complex. These estimates assume that there is nosignificant truncation or other processing of the protein subunits uponforming the toxin complex. What ever the true stochiometry of the toxincomplex turns out to be, it is clearly composed of multiple copies ofidentical subunits.

The exochitinase was identified by MALDI from what first appeared to bea homogeneous protein band migrating at a molecular mass consistent withthat of a truncated XptB1_(Xwi) (85 kDa). While the truncatedXptB1_(Xwi) in XTC-2 was positively identified, careful MALDI analysisenabled the discovery that the signal actually was derived from twoco-migrating proteins, XptB1_(Xwi) and an exochitinase.

This is the first demonstration of bioactivity from a Xenorhabdus toxincomplex containing co-purified XptD1_(Xwi) and exochitinase proteins,and might indicate an important role for this exochitinase in toxincomplex mode of action. The precise role of the exochitinase protein inproducing insecticidal activity is not known, nor do we know if theexochitinase being bound in the toxin complex has any biologicalactivity. Toxicity bioassay studies using Xeno Toxin Complex 1,recombinant XptA2_(Xwi) and other accessory proteins from Xenorhabdusand Photoharabdus, clearly demonstrate that an exochitinase enzyme isnot needed for expression of high levels of oral insecticidal activitywith these proteins. What is not known is if the chitinase acts in somemanner to increase insecticidal activity beyond what is expressed in itsabsence. Expression of exo and endochitinase genes in plants have beenshown to increase the activity of Bt toxins. (Ding et al., 1998; Regevet al., 1996). Such enzymatic activity might assist in breaking downchitin barriers in the peritrophic membrane separating the toxin fromits site of action in the gut of the insect. Being able to express theseproteins individually in heterologous systems and obtaining them inpurified form would allow more careful biochemical studies aimed atdetermining the individual role of each protein in producinginsecticidal activity.

XptD1_(Xwi) and XptE1_(Xwi) are two proteins very similar toXptA2_(Xwi). They may represent unique domains of XptA2_(Xwi) thatevolutionarily have been separated into two separate gene products.Having these proteins in purified form so that individual bioassays andbinding studies may be conducted may give clues to how these proteinsfunction in vivo. Such studies may also provide insight how theXptA2_(Xwi) protein might be mutated or tuncated and still retainbiological activity. The discovery of Xeno Toxin Complex 2 is animportant finding and we demonstrate here the activity of these proteinswith utility for insect control. It should be noted that XptD1_(Xwi) andXptE1_(Xwi) are about half the size of XptA2_(Xwi), and thus mightrepresent effective alternatives transgenes should they individuallypossess the biological potency and spectrum required for insectresistance product goal. They also provide information that can be usedto engineer XptA2_(Xwi) to make the protein smaller yet still retainactivity.

Proteins and toxins. The present invention provides easily administered,functional proteins. The invention also provides a method for deliveringinsecticidal toxins that are functionally active and effective againstmany orders of insects, preferably lepidopteran insects.

The subject invention provides new classes of toxins having advantageouspesticidal activities. One way to characterize these classes of toxinsand the polynucleotides that encode them is by defining a polynucleotideby its ability to hybridize, under a range of specified conditions, withan exemplified nucleotide sequence (the complement thereof and/or aprobe or probes derived from either strand) and/or by their ability tobe amplified by PCR using primers derived from the exemplifiedsequences.

There are a number of methods for obtaining the pesticidal toxins of theinstant invention. For example, antibodies to the pesticidal toxinsdisclosed and claimed herein can be used to identify and isolate othertoxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the toxins which are most constant and mostdistinct from other toxins. These antibodies can then be used tospecifically identify equivalent toxins with the characteristic activityby immunoprecipitation, enzyme linked immunosorbent assay (ELISA), orwestern blotting. Antibodies to the toxins disclosed herein, or toequivalent toxins, or to fragments of these toxins, can be readilyprepared using standard procedures. Toxins of the subject invention canbe obtained from a variety of sources/source microorganisms.

One skilled in the art would readily recognize that toxins (and genes)of the subject invention can be obtained from a variety of sources. Atoxin “from” or “obtainable from” the subject Xwi isolate means that thetoxin (or a similar toxin) can be obtained from Xwi or some othersource, such as another bacterial strain or a plant. For example, oneskilled in the art will readily recognize that, given the disclosure ofa bacterial gene and toxin, a plant can be engineered to produce thetoxin. Antibody preparations, nucleic acid probes (DNA and RNA), and thelike may be prepared using the polynucleotide and/or amino acidsequences disclosed herein and used to screen and recover other toxingenes from other (natural) sources.

Delivery of toxins. There are many other ways in which toxins can beincorporated into an insect's diet. For example, it is possible toadulterate the larval food source with the toxic protein by spraying thefood with a protein solution, as disclosed herein. Alternatively, thepurified protein could be genetically engineered into an otherwiseharmless bacterium, which could then be grown in culture, and eitherapplied to the food source or allowed to reside in the soil in an areain which insect eradication was desirable. Also, the protein could begenetically engineered directly into an insect food source. Forinstance, the major food source for many insect larvae is plantmaterial. Therefore the genes encoding toxins can be transferred toplant material so that said plant material expresses the toxin ofinterest.

Transfer of the functional activity to plant or bacterial systemstypically requires nucleic acid sequences, encoding the amino acidsequences for the toxins, integrated into a protein expression vectorappropriate to the host in which the vector will reside. One way toobtain a nucleic acid sequence encoding a protein with functionalactivity is to isolate the native genetic material from the bacterialspecies which produce the toxins, using information deduced from thetoxin's amino acid sequence, as disclosed herein. The native sequencescan be optimized for expression in plants, for example, as discussed inmore detail below. Optimized polynucleotide can also be designed basedon the protein sequence.

Polynucleotides and probes. The subject invention further providesnucleotide sequences that encode the toxins of the subject invention.The subject invention further provides methods of identifying andcharacterizing genes that encode pesticidal toxins. In one embodiment,the subject invention provides unique nucleotide sequences that areuseful as hybridization probes and/or primers for PCR techniques. Theprimers produce characteristic gene fragments that can be used in theidentification, characterization, and/or isolation of specific toxingenes. The nucleotide sequences of the subject invention encode toxinsthat are distinct from previously described toxins.

The polynucleotides of the subject invention can be used to formcomplete “genes” to encode proteins or peptides in a desired host cell.For example, as the skilled artisan would readily recognize, the subjectpolynucleotides can be appropriately placed under the control of apromoter in a host of interest, as is readily known in the art.

As the skilled artisan knows, DNA typically exists in a double-strandedform. In this arrangement, one strand is complementary to the otherstrand and vice versa. As DNA is replicated in a plant (for example),additional complementary strands of DNA are produced. The “codingstrand” is often used in the art to refer to the strand that binds withthe anti-sense strand. The mRNA is transcribed from the “anti-sense”strand of DNA. The “sense” or “coding” strand has a series of codons (acodon is three nucleotides that can be read as a three-residue unit tospecify a particular amino acid) that can be read as an open readingframe (ORF) to form a protein or peptide of interest. In order toproduce a protein in vivo, a strand of DNA is typically transcribed intoa complementary strand of mRNA which is used as the template for theprotein. Thus, the subject invention includes the use of the exemplifiedpolynucleotides shown in the attached sequence listing and/orequivalents including the complementary strands. RNA and PNA (peptidenucleic acids) that are functionally equivalent to the exemplified DNAare included in the subject invention.

In one embodiment of the subject invention, bacterial isolates can becultivated under conditions resulting in high multiplication of themicrobe. After treating the microbe to provide single-stranded genomicnucleic acid, the DNA can be contacted with the primers of the inventionand subjected to PCR amplification. Characteristic fragments oftoxin-encoding genes will be amplified by the procedure, thusidentifying the presence of the toxin-encoding gene(s).

Further aspects of the subject invention include genes and isolatesidentified using the methods and nucleotide sequences disclosed herein.The genes thus identified encode toxins active against pests.

Toxins and genes of the subject invention can be identified and obtainedby using oligonucleotide probes, for example. These probes aredetectable nucleotide sequences which may be detectable by virtue of anappropriate label or may be made inherently fluorescent as described inInternational Application No. WO 93/16094. The probes (and thepolynucleotides of the subject invention) may be DNA, RNA, or PNA. Inaddition to adenine (A), cytosine (C), guanine (G), thymine (T), anduracil (U; for RNA molecules), synthetic probes (and polynucleotides) ofthe subject invention can also have inosine (a neutral base capable ofpairing with all four bases; sometimes used in place of a mixture of allfour bases in synthetic probes). Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “n” is used generically, “n”can be G, A, T, C, or inosine. Ambiguity codes as used herein are inaccordance with standard IUPAC naming conventions as of the filing ofthe subject application (for example, R means A or G, Y means C or T,etc.).

As is well known in the art, if a probe molecule hybridizes with anucleic acid sample, it can be reasonably assumed that the probe andsample have substantial homology/similarity/identity. Preferably,hybridization of the polynucleotide is first conducted followed bywashes under conditions of low, moderate, or high stringency bytechniques well-known in the art, as described in, for example, Keller,G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y.,pp. 169-170. For example, as stated therein, low stringency conditionscan be achieved by first washing with 2×SSC (Standard SalineCitrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at roomtemperature. Two washes are typically performed. Higher stringency canthen be achieved by lowering the salt concentration and/or by raisingthe temperature. For example, the wash described above can be followedby two washings with 0.1×SSC/0.1% SDS for 15 minutes each at roomtemperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30minutes each at 55E C. These temperatures can be used with otherhybridization and wash protocols set forth herein and as would be knownto one skilled in the art (SSPE can be used as the salt instead of SSC,for example). The 2×SSC/0.1% SDS can be prepared by adding 50 ml of20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSC can be prepared bycombining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), andwater to 1 liter, followed by adjusting pH to 7.0 with 10 N NaOH. 10%SDS can be prepared by dissolving 10 g of SDS in 50 ml of autoclavedwater, diluting to 100 ml, and aliquotting.

Detection of the probe provides a means for determining in a knownmanner whether hybridization has been maintained. Such a probe analysisprovides a rapid method for identifying toxin-encoding genes of thesubject invention. The nucleotide segments which are used as probesaccording to the invention can be synthesized using a DNA synthesizerand standard procedures. These nucleotide sequences can also be used asPCR primers to amplify genes of the subject invention.

Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide exemplifiedherein.

Duplex formation and stability depend on substantial complementarilybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

PCR technology. Polymerase Chain Reaction (PCR) is a repetitive,enzymatic, primed synthesis of a nucleic acid sequence. This procedureis well known and commonly used by those skilled in this art (seeMullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki,Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn,Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification ofP-Globin Genomic Sequences and Restriction Site Analysis for Diagnosisof Sickle Cell Anemia,” Science 230:1350-1354). PCR is based on theenzymatic amplification of a DNA fragment of interest that is flanked bytwo oligonucleotide primers that hybridize to opposite strands of thetarget sequence. The primers are oriented with the 3′ ends pointingtowards each other. Repeated cycles of heat denaturation of thetemplate, annealing of the primers to their complementary sequences, andextension of the annealed primers with a DNA polymerase result in theamplification of the segment defined by the 5′ ends of the PCR primers.The extension product of each primer can serve as a template for theother primer, so each cycle essentially doubles the amount of DNAfragment produced in the previous cycle. This results in the exponentialaccumulation of the specific target fragment, up to several million-foldin a few hours. By using a thermostable DNA polymerase such as Taqpolymerase, isolated from the thermophilic bacterium Thermus aquaticus,the amplification process can be completely automated. Other enzymeswhich can be used are known to those skilled in the art.

The DNA sequences of the subject invention can be used as primers forPCR amplification. In performing PCR amplification, a certain degree ofmismatch can be tolerated between primer and template. Therefore,mutations, deletions, and insertions (especially additions ofnucleotides to the 5N end) of the exemplified primers fall within thescope of the subject invention. Mutations, insertions, and deletions canbe produced in a given primer by methods known to an ordinarily skilledartisan.

Modification of genes and toxins. The genes and toxins useful accordingto the subject invention include not only the specifically exemplifiedfull-length sequences, but also portions, segments and/or fragments(including internal and/or terminal deletions compared to thefull-length molecules) of these sequences, variants, mutants, chimerics,and fusions thereof Proteins of the subject invention can havesubstituted amino acids so long as they retain the characteristicpesticidal/ functional activity of the proteins specifically exemplifiedherein. “Variant” genes have nucleotide sequences that encode the sametoxins or equivalent toxins having pesticidal activity equivalent to anexemplified protein. The terms “variant proteins” and “equivalenttoxins” refer to toxins having the same or essentially the samebiological/functional activity against the target pests and equivalentsequences as the exemplified toxins. As used herein, reference to an“equivalent” sequence refers to sequences having amino acidsubstitutions, deletions, additions, or insertions which improve or donot adversely affect pesticidal activity. Fragments retaining pesticidalactivity are also included in this definition. Fragments and otherequivalents that retain the same or similar function, or “toxinactivity,” as a corresponding fragment of an exemplified toxin arewithin the scope of the subject invention. Changes, such as amino acidsubstitutions or additions, can be made for a variety of purposes, suchas increasing (or decreasing) protease stability of the protein (withoutmaterially/substantially decreasing the functional activity of thetoxin).

Equivalent toxins and/or genes encoding these equivalent toxins can beobtained/derived from wild-type or recombinant bacteria and/or fromother wild-type or recombinant organisms using the teachings providedherein. Other Bacillus, Paenibacillus, Photorhabdus, and Xenorhabdusspecies, for example, can be used as source isolates.

Variations of genes may be readily constructed using standard techniquesfor making point mutations, for example. In addition, U.S. Pat. No.5,605,793, for example, describes methods for generating additionalmolecular diversity by using DNA reassembly after random fragmentation.Variant genes can be used to produce variant proteins; recombinant hostscan be used to produce the variant proteins. Using these “geneshuffling” techniques, equivalent genes and proteins can be constructedthat comprise any 5, 10, or 20 contiguous residues (amino acid ornucleotide) of any sequence exemplified herein. As one skilled in theart knows, the gene shuffling techniques can be adjusted to obtainequivalents having, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438,439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466,467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494,495, 496, 497, 498, 499, or 500 contiguous residues (amino acid ornucleotide), corresponding to a segment (of the same size) in any of theexemplified sequences (or the complements (full complements) thereof).Similarly sized segments, especially those for conserved regions, canalso be used as probes and/or primers.

Fragments of full-length genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes which encode active fragments may be obtained using avariety of restriction enzymes. Proteases may be used to directly obtainactive fragments of these toxins.

It is within the scope of the invention as disclosed herein that toxinsmay be truncated and still retain functional activity. By “truncatedtoxin” is meant that a portion of a toxin protein may be cleaved and yetstill exhibit activity after cleavage. Cleavage can be achieved byproteases inside or outside of the insect gut. Furthermore, effectivelycleaved proteins can be produced using molecular biology techniqueswherein the DNA bases encoding said toxin are removed either throughdigestion with restriction endonucleases or other techniques availableto the skilled artisan. After truncation, said proteins can be expressedin heterologous systems such as E coli, baculoviruses, plant-based viralsystems, yeast and the like and then placed in insect assays asdisclosed herein to determine activity. It is well-known in the art thattruncated toxins can be successfully produced so that they retainfunctional activity while having less than the entire, full-lengthsequence. It is well known in the art that B. t. toxins can be used in atruncated (core toxin) form. See, e.g., Adang el al, Gene 36:289-300(1985), “Characterized full-length and truncated plasmid clones of thecrystal protein of Bacillus thuringiensis subsp kurstaki HD-73 and theirtoxicity to Manduca sexta.” There are other examples of truncatedproteins that retain insecticidal activity, including the insectjuvenile hormone esterase (U.S. Pat. No. 5,674,485 to the Regents of theUniversity of California). As used herein, the term “toxin” is alsomeant to include functionally active truncations.

Certain toxins of the subject invention have been specificallyexemplified herein. As these toxins are merely exemplary of the toxinsof the subject invention, it should be readily apparent that the subjectinvention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid similarity (and/or homology) with an exemplified toxin.The amino acid identity will typically be greater than 60%, preferablygreater than 75%, more preferably greater than 80%, even more preferablygreater than 90%, and can be greater than 95%. Preferred polynucleotidesand proteins of the subject invention can also be defined in terms ofmore particular identity and/or similarity ranges. For example, theidentity and/or similarity can be 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as comparedto a sequence exemplified herein.

The amino acid homology/similarity/identity will be highest in criticalregions of the toxin which account for biological activity or areinvolved in the determination of three-dimensional configuration whichis ultimately responsible for the biological activity. In this regard,certain amino acid substitutions are acceptable and can be expected tobe tolerated. For example, these substitutions can be in regions of theprotein that are not critical to activity. Analyzing the crystalstructure of a protein, and software-based protein structure modeling,can be used to identify regions of a protein that can be modified (usingsite-directed mutagenesis, shuffling, etc.) to actually change theproperties and/or increase the functionality of the protein.

Various properties and three-dimensional features of the protein canalso be changed without adversely affecting the toxinactivity/functionality of the protein. Conservative amino acidsubstitutions can be expected to be tolerated/to not adversely affectthe three-dimensional configuration of the molecule. Amino acids can beplaced in the following classes: non-polar, uncharged polar, basic, andacidic. Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution is not adverse tothe biological activity of the compound. Table 1 provides a listing ofexamples of amino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the functional/biological activity of the toxin.

Because of the degeneracy/redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate alternative DNA sequences that encode the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention.

Optimization of sequence for expression in plants. To obtain highexpression of heterologous genes in plants it may be preferred toreengineer said genes so that they are more efficiently expressed in(the cytoplasm of) plant cells. Maize is one such plant where it may bepreferred to re-design the heterologous gene(s) prior to transformationto increase the expression level thereof in said plant. Therefore, anadditional step in the design of genes encoding a bacterial toxin isreengineering of a heterologous gene for optimal expression.

One reason for the reengineering of a bacterial toxin for expression inmaize is due to the non-optimal G+C content of the native gene. Forexample, the very low G+C content of many native bacterial gene(s) (andconsequent skewing towards high A+T content) results in the generationof sequences mimicking or duplicating plant gene control sequences thatare known to be highly A+T rich. The presence of some A+T-rich sequenceswithin the DNA of gene(s) introduced into plants (e.g., TATA box regionsnormally found in gene promoters) may result in aberrant transcriptionof the gene(s). On the other hand, the presence of other regulatorysequences residing in the transcribed MRNA (e.g., polyadenylation signalsequences (AAUAAA), or sequences complementary to small nuclear RNAsinvolved in pre-mRNA splicing) may lead to RNA instability. Therefore,one goal in the design of genes encoding a bacterial toxin for maizeexpression, more preferably referred to as plant optimized gene(s), isto generate a DNA sequence having a higher G+C content, and preferablyone close to that of maize genes coding for metabolic enzymes. Anothergoal in the design of the plant optimized gene(s) encoding a bacterialtoxin is to generate a DNA sequence in which the sequence modificationsdo not hinder translation.

The table below (Table 2) illustrates how high the G+C content is inmaize. For the data in Table 2, coding regions of the genes wereextracted from GenBank (Release 71) entries, and base compositions werecalculated using the MacVector.TM. program (IBI, New Haven, Conn.).Intron sequences were ignored in the calculations.

Due to the plasticity afforded by the redundancy/degeneracy of thegenetic code (i.e., some amino acids are specified by more than onecodon), evolution of the genomes in different organisms or classes oforganisms has resulted in differential usage of redundant codons. This“codon bias” is reflected in the mean base composition of protein codingregions. For example, organisms with relatively low G+C contents utilizecodons having A or T in the third position of redundant codons, whereasthose having higher G+C contents utilize codons having G or C in thethird position. It is thought that the presence of “minor” codons withina mRNA may reduce the absolute translation rate of that mRNA, especiallywhen the relative abundance of the charged tRNA corresponding to theminor codon is low. An extension of this is that the diminution oftranslation rate by individual minor codons would be at least additivefor multiple minor codons. Therefore, mRNAs having high relativecontents of minor codons would have correspondingly low translationrates. This rate would be reflected by subsequent low levels of theencoded protein.

In engineering genes encoding a bacterial toxin for maize (or otherplant, such as cotton or soybean) expression, the codon bias of theplant has been determined. The codon bias for maize is the statisticalcodon distribution that the plant uses for coding its proteins and thepreferred codon usage is shown in Table 3. After determining the bias,the percent frequency of the codons in the gene(s) of interest isdetermined. The primary codons preferred by the plant should bedetermined as well as the second and third choice of preferred codons.Afterwards, the amino acid sequence of the bacterial toxin of interestis reverse translated so that the resulting nucleic acid sequence codesfor exactly the same protein as the native gene wanting to beheterologously expressed. The new DNA sequence is designed using codonbias information so that it corresponds to the most preferred codons ofthe desired plant. The new sequence is then analyzed for restrictionenzyme sites that might have been created by the modification. Theidentified sites are further modified by replacing the codons withsecond or third choice preferred codons. Other sites in the sequencewhich could affect transcription or translation of the gene of interestare the exon:intron junctions (5′ or 3′), poly A addition signals, orRNA polymerase termination signals. The sequence is further analyzed andmodified to reduce the frequency of TA or GC doublets. In addition tothe doublets, G or C sequence blocks that have more than about fourresidues that are the same can affect transcription of the sequence.Therefore, these blocks are also modified by replacing the codons offirst or second choice, etc. with the next preferred codon of choice.

TABLE 2 Compilation of G + C contents of protein coding regions of maizegenes Protein Class.sup.a Range % G + C Mean % G + C.sup.b MetabolicEnzymes (76) 44.4-75.3 59.0 (.+−.8.0) Structural Proteins (18) 48.6-70.563.6 (.+−.6.7) Regulatory Proteins (5) 57.2-68.8 62.0 (.+−.4.9)Uncharacterized Proteins (9) 41.5-70.3 64.3 (.+−.7.2) All Proteins (108)44.4-75.3 60.8 (.+−.5.2) .sup.a Number of genes in class given inparentheses. .sup.b Standard deviations given in parentheses. .sup.cCombined groups mean ignored in mean calculation

It is preferred that the plant optimized gene(s) encoding a bacterialtoxin contain about 63% of first choice codons, between about 22% toabout 37% second choice codons, and between about 15% to about 0% thirdchoice codons, wherein the total percentage is 100%. Most preferred theplant optimized gene(s) contains about 63% of first choice codons, atleast about 22% second choice codons, about 7.5% third choice codons,and about 7.5% fourth choice codons, wherein the total percentage is100%. The preferred codon usage for engineering genes for maizeexpression are shown in Table 3. The method described above enables oneskilled in the art to modify gene(s) that are foreign to a particularplant so that the genes are optimally expressed in plants. The method isfurther illustrated in PCT application WO 97/13402.

In order to design plant optimized genes encoding a bacterial toxin, theamino acid sequence of said protein is reverse translated into a DNAsequence utilizing a non-redundant genetic code established from a codonbias table compiled for the gene sequences for the particular plant, asshown in Table 2. The resulting DNA sequence, which is completelyhomogeneous in codon usage, is further modified to establish a DNAsequence that, besides having a higher degree of codon diversity, alsocontains strategically placed restriction enzyme recognition sites,desirable base composition, and a lack of sequences that might interferewith transcription of the gene, or translation of the product mRNA.

TABLE 3 Preferred amino acid codons for proteins expressed in maizeAmino Acid Codon* Alanine GCC/GCG Cysteine TGC/TGT Aspartic Acid GAC/GATGlutamic Acid GAG/GAA Phenylalanine TTC/TTT Glycine GGC/GGG HistidineCAC/CAT Isoleucine ATC/ATT Lysine AAG/AAA Leucine CTG/CTC Methionine ATGAsparagine AAC/AAT Proline CCG/CCA Glutamine CAG/CAA Arginine AGG/CGCSerine AGC/TCC Threonine ACC/ACG Valine GTG/GTC Tryptophan TGG TryrosineTAC/TAT Stop TGA/TAG *The first and second preferred codons for maize.

Thus, synthetic genes that are functionally equivalent to thetoxins/genes of the subject invention can be used to transform hosts,including plants. Additional guidance regarding the production ofsynthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.

In some cases, especially for expression in plants, it can beadvantageous to use truncated genes that express truncated proteins.Höfte et al. 1989, for example, discussed in the Background Sectionabove, discussed protoxin and core toxin segments of B. t. toxins.Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% of the full-length toxin.

Transgenic hosts. The toxin-encoding genes of the subject invention canbe introduced into a wide variety of microbial or plant hosts. Inpreferred embodiments, transgenic plant cells and plants are used.Preferred plants (and plant cells) are corn, maize, and cotton.

In preferred embodiments, expression of the toxin gene results, directlyor indirectly, in the intracellular production (and maintenance) of thepesticide proteins. Plants can be rendered insect-resistant in thismanner. When transgenic/recombinant/transformed/transfected host cells(or contents thereof) are ingested by the pests, the pests will ingestthe toxin. This is the preferred manner in which to cause contact of thepest with the toxin. The result is control (killing or making sick) ofthe pest. Sucking pests can also be controlled in a similar manner.Alternatively, suitable microbial hosts, e.g., Pseudomonas such as P.fluorescens, can be applied where target pests are present; the microbescan proliferate there, and are ingested by the target pests. The microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin and stabilize the cell. The treated cell, whichretains the toxic activity, can then be applied to the environment ofthe target pest.

Where the toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, certain host microbes should be used. Microorganism hosts areselected which are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops ofinterest. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetohacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharornyces, Cryptococcus,Kluyvetomyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomnyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Also of interest arepigmented microorganisms.

Insertion of genes to form transgenic hosts. One aspect of the subjectinvention is the transformation/transfection of plants, plant cells, andother host cells with polynucleotides of the subject invention thatexpress proteins of the subject invention. Plants transformed in thismanner can be rendered resistant to attack by the target pest(s).

A wide variety of methods are available for introducing a gene encodinga pesticidal protein into the target host under conditions that allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867.

For example, a large number of cloning vectors comprising a replicationsystem in E. coli and a marker that permits selection of the transformedcells are available for preparation for the insertion of foreign genesinto higher plants. The vectors comprise, for example, pBR322, pUCseries, M13 mp series, pACYC184, etc. Accordingly, the sequence encodingthe toxin can be inserted into the vector at a suitable restrictionsite. The resulting plasmid is used for transformation into E. coli. TheE. coli cells are cultivated in a suitable nutrient medium, thenharvested and lysed. The plasmid is recovered. Sequence analysis,restriction analysis, electrophoresis, and other biochemical-molecularbiological methods are generally carried out as methods of analysis.After each manipulation, the DNA sequence used can be cleaved and joinedto the next DNA sequence. Each plasmid sequence can be cloned in thesame or other plasmids. Depending on the method of inserting desiredgenes into the plant, other DNA sequences may be necessary. If, forexample, the Ti or Ri plasmid is used for the transformation of theplant cell, then at least the right border, but often the right and theleft border of the Ti or Ri plasmid T-DNA, has to be joined as theflanking region of the genes to be inserted. The use of T-DNA for thetransformation of plant cells has been intensively researched anddescribed in EP 120 516; Hoekema (1985) In: The Binary Plant VectorSystem, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraleyel al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J.4:277-287.

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and inAgrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters el al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

In some preferred embodiments of the invention, genes encoding thebacterial toxin are expressed from transcriptional units inserted intothe plant genome. Preferably, said transcriptional units are recombinantvectors capable of stable integration into the plant genome and enableselection of transformed plant lines expressing mRNA encoding theproteins.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there (and does not come out again). It normallycontains a selection marker that confers on the transformed plant cellsresistance to a biocide or an antibiotic, such as kanamycin, G418,bleomycin, hygromycin, or chloramphenicol, inter alia. The individuallyemployed marker should accordingly permit the selection of transformedcells rather than cells that do not contain the inserted DNA. Thegene(s) of interest are preferably expressed either by constitutive orinducible promoters in the plant cell. Once expressed, the mRNA istranslated into proteins, thereby incorporating amino acids of interestinto protein. The genes encoding a toxin expressed in the plant cellscan be under the control of a constitutive promoter; a tissue-specificpromoter, or an inducible promoter.

Several techniques exist for introducing foreign recombinant vectorsinto plant cells, and for obtaining plants that stably maintain andexpress the introduced gene. Such techniques include the introduction ofgenetic material coated onto microparticles directly into cells (U.S.Pat. Nos. 4,945,050 to Cornell and 5,141,131 to DowElanco, now DowAgroSciences, LLC). In addition, plants may be transformed usingAgrobacterium technology, see U.S. Pat. No. 5,177,010 to University ofToledo; 5,104,310 to Texas A&M; European Patent Application 0131624B1;European Patent Applications 120516, 159418B1 and 176,112 toSchilperoot; U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and4,940,838 and 4,693,976 to Schilperoot; European Patent Applications116718, 290799, 320500 all to Max Planck; European Patent Applications604662 and 627752, and U.S. Pat. No. 5,591,616, to Japan Tobacco;European Patent Applications 0267159 and 0292435, and U.S. Pat. No.5,231,019, all to Ciba Geigy, now Novartis; U.S. Pat. Nos. 5,463,174 and4,762,785, both to Calgene; and U.S. Pat. Nos. 5,004,863 and 5,159,135,both to Agracetus. Other transformation technology includes whiskerstechnology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca.Electroporation technology has also been used to transform plants. SeeWO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and5,384,253, both to Dekalb; and WO 92/09696 and WO 93/21335, both toPlant Genetic Systems. Furthermore, viral vectors can also be used toproduce transgenic plants expressing the protein of interest. Forexample, monocotyledonous plant can be transformed with a viral vectorusing the methods described in U.S. Pat. No. 5,569,597 to Mycogen PlantScience and Ciba-Giegy, now Novartis, as well as U.S. Pat. Nos.5,589,367 and 5,316,931, both to Biosource.

As mentioned previously, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod which provides for efficient transformation may be employed. Forexample, various methods for plant cell transformation are describedherein and include the use of Ti or Ri-plasmids and the like to performAgrobacterium mediated transformation. In many instances, it will bedesirable to have the construct used for transformation bordered on oneor both sides by T-DNA borders, more specifically the right border. Thisis particularly useful when the construct uses Agrobacterium tumefaciensor Agrobacterium rhizogenes as a mode for transformation, although T-DNAborders may find use with other modes of transformation. WhereAgrobacterium is used for plant cell transformation, a vector may beused which may be introduced into the host for homologous recombinationwith T-DNA or the Ti or Ri plasmid present in the host. Introduction ofthe vector may be performed via electroporation, tri-parental mating andother techniques for transforming gram-negative bacteria which are knownto those skilled in the art. The manner of vector transformation intothe Agrobacterium host is not critical to this invention. The Ti or Riplasmid containing the T-DNA for recombination may be capable orincapable of causing gall formation, and is not critical to saidinvention so long as the vir genes are present in said host.

In some cases where Agrobacterium is used for transformation, theexpression construct being within the T-DNA borders will be insertedinto a broad spectrum vector such as pRK2 or derivatives thereof asdescribed in Ditta et al., (PNAS USA(1980) 77:7347-7351 and EPO 0 120515, which are incorporated herein by reference. Included within theexpression construct and the T-DNA will be one or more markers asdescribed herein which allow for selection of transformedAgrobacteriumand transformed plant cells. The particular marker employedis not essential to this invention, with the preferred marker dependingon the host and construction used.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime to allow transformation thereof. After transformation, theAgrobacteria are killed by selection with the appropriate antibiotic andplant cells are cultured with the appropriate selective medium. Oncecalli are formed, shoot formation can be encouraged by employing theappropriate plant hormones according to methods well known in the art ofplant tissue culturing and plant regeneration. However, a callusintermediate stage is not always necessary. After shoot formation, saidplant cells can be transferred to medium which encourages root formationthereby completing plant regeneration. The plants may then be grown toseed and said seed can be used to establish future generations.Regardless of transformation technique, the gene encoding a bacterialtoxin is preferably incorporated into a gene transfer vector adapted toexpress said gene in a plant cell by including in the vector a plantpromoter regulatory element, as well as 3′ non-translatedtranscriptional termination regions such as Nos and the like.

In addition to numerous technologies for transforming plants, the typeof tissue which is contacted with the foreign genes may vary as well.Such tissue would include but would not be limited to embryogenictissue, callus tissue types I, II, and III, hypocotyl, meristem, roottissue, tissues for expression in phloem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques described herein.

As mentioned above, a variety of selectable markers can be used, ifdesired. Preference for a particular marker is at the discretion of theartisan, but any of the following selectable markers may be used alongwith any other gene not listed herein which could function as aselectable marker. Such selectable markers include but are not limitedto aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II)which encodes resistance to the antibiotics kanamycin, neomycin andG418, as well as those genes which encode for resistance or tolerance toglyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos);imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such aschlorsulfuron; bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used with or without aselectable marker. Reporter genes are genes which are typically notpresent in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property.Examples of such genes are provided in K. Wising et al. Ann. Rev.Genetics, 22, 421 (1988). Preferred reporter genes include thebeta-glucuronidase (GUS) of the uidA locus of E. coli, thechloramphenicol acetyl transferase gene from Tn9 of E. coli, the greenfluorescent protein from the bioluminescent jellyfish Aequorea victoria,and the luciferase genes from firefly Photinus pyralis. An assay fordetecting reporter gene expression may then be performed at a suitabletime after said gene has been introduced into recipient cells. Apreferred such assay entails the use of the gene encodingbeta-glucuronidase (GUS) of the uidA locus of E. coli as described byJefferson el al., (1987 Biochem. Soc. Trans. 15, 17-19) to identifytransformed cells.

In addition to plant promoter regulatory elements, promoter regulatoryelements from a variety of sources can be used efficiently in plantcells to express foreign genes. For example, promoter regulatoryelements of bacterial origin, such as the octopine synthase promoter,the nopaline synthase promoter, the mannopine synthase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S and19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No.6,166,302, especially Example 7E) and the like may be used. Plantpromoter regulatory elements include but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter,heat-shock promoters, and tissue specific promoters. Other elements suchas matrix attachment regions, scaffold attachment regions, introns,enhancers, polyadenylation sequences and the like may be present andthus may improve the transcription efficiency or DNA integration. Suchelements may or may not be necessary for DNA function, although they canprovide better expression or functioning of the DNA by affectingtranscription, mRNA stability, and the like. Such elements may beincluded in the DNA as desired to obtain optimal performance of thetransformed DNA in the plant. Typical elements include but are notlimited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coatprotein leader sequence, the maize streak virus coat protein leadersequence, as well as others available to a skilled artisan. Constitutivepromoter regulatory elements may also be used thereby directingcontinuous gene expression in all cells types and at all times (e.g.,actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoterregulatory elements are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g., zein, oleosin,napin, ACP, globulin and the like) and these may also be used.

Promoter regulatory elements may also be active during a certain stageof the plant's development as well as active in plant tissues andorgans. Examples of such include but are not limited to pollen-specific,embryo-specific, corn-silk-specific, cotton-fiber-specific,root-specific, seed-endosperm-specific promoter regulatory elements andthe like. Under certain circumstances it may be desirable to use aninducible promoter regulatory element, which is responsible forexpression of genes in response to a specific signal, such as: physicalstimulus (heat shock genes), light (RUBP carboxylase), hormone (Em),metabolites, chemical, and stress. Other desirable transcription andtranslation elements that function in plants may be used. Numerousplant-specific gene transfer vectors are known in the art.

Standard molecular biology techniques may be used to clone and sequencethe toxins described herein. Additional information may be found inSambrook, J., Fritsch, E. F., and Maniatis, T. (1989), MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, which isincorporated herein by reference.

Resistance Management. With increasing commercial use of insecticidalproteins in transgenic plants, one consideration is resistancemanagement. That is, there are numerous companies using Bacillusthuringiensis toxins in their products, and there is concern aboutinsects developing resistance to B. t. toxins. One strategy for insectresistance management would be to combine the TC toxins produced byXenorhabdus, Photorhabdus, and the like with toxins such as B. t.crystal toxins, soluble insecticidal proteins from Bacillus stains (see,e.g., WO 98/18932 and WO 99/57282), or other insect toxins. Thecombinations could be formulated for a sprayable application or could bemolecular combinations. Plants could be transformed with bacterial genesthat produce two or more different insect toxins (see, e.g., Gould, 38Bioscience 26-33 (1988) and U.S. Pat. No. 5,500,365; likewise, EuropeanPatent Application 0 400 246 A1 and U.S. Pats. 5,866,784; 5,908,970; and6,172,281 also describe transformation of a plant with two B. t. crystaltoxins). Another method of producing a transgenic plant that containsmore than one insect resistant gene would be to first produce twoplants, with each plant containing an insect resistance gene. Theseplants could then be crossed using traditional plant breeding techniquesto produce a plant containing more than one insect resistance gene.Thus, it should be apparent that the phrase “comprising apolynucleotide” as used herein means at least one polynucleotide (andpossibly more, contiguous or not) unless specifically indicatedotherwise.

Formulations and Other Delivery Systems. Formulated bait granulescontaining spores and/or crystals of the subject Paenibacillus isolate,or recombinant microbes comprising the genes obtainable from the isolatedisclosed herein, can be applied to the soil. Formulated product canalso be applied as a seed-coating or root treatment or total planttreatment at later stages of the crop cycle. Plant and soil treatmentsof cells may be employed as wettable powders, granules or dusts, bymixing with various inert materials, such as inorganic minerals(phyllosilicates, carbonates, sulfates, phosphates, and the like) orbotanical materials (powdered corncobs, rice hulls, walnut shells, andthe like). The formulations may include spreader-sticker adjuvants,stabilizing agents, other pesticidal additives, or surfactants. Liquidformulations may be aqueous-based or non-aqueous and employed as foams,gels, suspensions, emulsifiable concentrates, or the like. Theingredients may include Theological agents, surfactants, emulsifiers,dispersants, or polymers.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1-95% by weight of the pesticide while the liquid formulations willgenerally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g.,soil and foliage, by spraying, dusting, sprinkling, or the like.

Another delivery scheme is the incorporation of the genetic material oftoxins into a baculovirus vector. Baculoviruses infect particular insecthosts, including those desirably targeted with the toxins. Infectiousbaculovirus harboring an expression construct for the toxins could beintroduced into areas of insect infestation to thereby intoxicate orpoison infected insects.

Insect viruses, or baculoviruses, are known to infect and adverselyaffect certain insects. The affect of the viruses on insects is slow,and viruses do not immediately stop the feeding of insects. Thus,viruses are not viewed as being optimal as insect pest control agents.However, combining the toxin genes into a baculovirus vector couldprovide an efficient way of transmitting the toxins. In addition, sincedifferent baculoviruses are specific to different insects, it may bepossible to use a particular toxin to selectively target particularlydamaging insect pests. A particularly useful vector for the toxins genesis the nuclear polyhedrosis virus. Transfer vectors using this virushave been described and are now the vectors of choice for transferringforeign genes into insects. The virus-toxin gene recombinant may beconstructed in an orally transmissible form. Baculoviruses normallyinfect insect victims through the mid-gut intestinal mucosa. The toxingene inserted behind a strong viral coat protein promoter would beexpressed and should rapidly kill the infected insect.

In addition to an insect virus or baculovirus or transgenic plantdelivery system for the protein toxins of the present invention, theproteins may be encapsulated using Bacillus thuringiensis encapsulationtechnology such as but not limited to U.S. Pat. Nos. 4,695,455;4,695,462; 4,861,595 which are all incorporated herein by reference.Another delivery system for the protein toxins of the present inventionis formulation of the protein into a bait matrix, which could then beused in above and below ground insect bait stations. Examples of suchtechnology include but are not limited to PCT Patent Application WO93/23998, which is incorporated herein by reference.

Plant RNA viral based systems can also be used to express bacterialtoxin. In so doing, the gene encoding a toxin can be inserted into thecoat promoter region of a suitable plant virus which will infect thehost plant of interest. The toxin can then be expressed thus providingprotection of the plant from insect damage. Plant RNA viral basedsystems are described in U.S. Pat. No. 5,500,360 to Mycogen PlantSciences, Inc. and U.S. Pat. Nos. 5,316,931 and 5,589,367 to BiosourceGenetics Corp.

In addition to producing a transformed plant, there are other deliverysystems where it may be desirable to reengineer the bacterial gene(s).For example, a protein toxin can be constructed by fusing together amolecule attractive to insects as a food source with a toxin. Afterpurification in the laboratory such a toxic agent with “built-in” baitcould be packaged inside standard insect trap housings.

Mutants. Mutants of the Xenorhabdus Xwi isolate of the invention can bemade by procedures that are well known in the art. For example,asporogenous mutants can be obtained through ethylmethane sulfonate(EMS) mutagenesis of an isolate. The mutants can be made usingultraviolet light and nitrosoguanidine by procedures well known in theart.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

1. An isolated polynucleotide of SEQ ID NO:3.
 2. A DNA moleculecomprising a nucleic acid sequence encoding the polypeptide of SEQ IDNO: 4 operatively associated with a heterologous promoter.
 3. Atransgenic microorganism comprising the DNA of claim
 2. 4. A transgenicplant comprising the DNA of claim
 2. 5. A transgenic plant cellcomprising the DNA of claim
 2. 6. A transgenic seed comprising the plantcell of claim
 5. 7. An isolated polynucleotide that encodes thepolypeptide of SEQ ID NO: 4 (XptE1_(Xwi)).
 8. A purified bacterial cellcomprising the isolated polynucleotide of claim
 7. 9. An isolatedpolynucleotide that encodes a protein at least 99% identical to SEQ IDNO: 4.